Quantification of 2D Gel Western Blot Images

from Human Tumor Samples

Nancy Kendrick, Matt Hoelter, Andrew Koll and Jon Johansen

Kendrick Labs, Inc, Madison, WI

www.kendricklabs.com

Introduction

Kendrick Labs, Inc is a contract research organization; our clients are mostly

scientists in pharmaceutical companies and academia. The goal of this project is to

develop a test that will be useful for cancer researchers studying signaling pathways.

Previously, we have shown that *CA-2DE, in combination with **ECL western blotting

can detect and identify low abundance tyrosine kinase (TK) proteins.

However, these results consist of western blot pictures. In order to be useful, the

results must be quantified and presented in a table as numbers. This talk is about our

preliminary quantification work.

• Note: Many TKs are difficult to dissolve. Only one buffer works well: ***SDS!

Our carrier ampholine 2D system is compatible with SDS.

• SDS strongly interferes with mass spectrometry and IPG strip 2D; most core labs avoid it. They use

SDS-free buffers and centrifuge out “cellular debris” containing important proteins. Kendrick Labs

offers a unique method for visualizing TKs.

2

*CA-2DE: carrier ampholine 2D electrophoresis, **ECL: enhanced chemiluminescence, ***SDS: sodium dodecyl sulfate

3

Receptor tyrosine kinase mechanism

Unphosphorylated RTKs are

present in many tissues.

If no tyrosine phosphorylation,

then no RTK activity!

RTKs are large trans-membrane proteins.

Ligand binding triggers dimerization, leading to

trans-phosphorylation of tyrosines on the

cytoplasmic chains (red circles with P)

Then, cytoplasmic proteins with affinity for

specific phosphotyrosines relocate to the

membrane, become activated, and trigger

cascades of cell growth reactions.

For more information see: Biology of Cancer,

2nd Ed. by Robert Weinberg, especially the EGFR

movie on the CD.

Pharma companies have already developed inhibitors for several TKs involved in lung cancer. It’s a hot research topic.

4

References

1. Bronte, G., et al., Are erlotinib and gefitinib interchangeable, opposite or complementary for non-small cell lung

cancer treatment? Critical reviews in oncology/hematology, 2014. 89(2): p. 300-13.

2. Roengvoraphoj, M., et al., Epidermal growth factor receptor tyrosine kinase inhibitors as initial therapy for non-small

cell lung cancer: focus on epidermal growth factor receptor mutation testing and mutation-positive patients. Cancer

treatment reviews, 2013. 39(8): p. 839-50.

3. Qian, H., et al., The efficacy and safety of crizotinib in the treatment of anaplastic lymphoma kinase-positive non-

small cell lung cancer: a meta-analysis of clinical trials. BMC Cancer, 2014. 14: p. 683.

4. Bria, E., S. Pilotto, and G. Tortora, Sorafenib for lung cancer: is the "Battle" still open? Expert Opin Investig Drugs,

2012. 21(10): p. 1445-8.

5. Scagliotti, G.V., S. Novello, and J. von Pawel, The emerging role of MET/HGF inhibitors in oncology. Cancer Treat

Rev, 2013. 39(7): p. 793-801.

6. Gold, K.A., et al., A Phase I/II Study Combining Erlotinib and Dasatinib for Non-Small Cell Lung Cancer. Oncologist,

2014. 19(10): p. 1040-1.

Tyrosine Kinase Protein Abbrevation

Molecular

weight Inhibitor Ref

Epidermal Growth Factor Receptor EGFR 170,000 Several [1,2]

Anaplastic Lymphoma Kinase ALK 176,000 Crizotinib [3]

Platelet Derived Growth Factor Receptor PDGFR 175,000 Sorafenib [4]

Hepatocyte Growth Factor Receptor cMET, HGFR 160,000 Several [5]

SRC (Cytoplasmic TK) SRC 60,000 Dasatinib [6]

Receptor tyrosine kinases tend to be around the same molecular weight and don’t resolve

on 1D gels. They’re hard to measure. Genomic tests are only partially successful. A 2DE

test that directly measures protein TK drivers should be useful to pharma companies.

Previously, we have identified active EGFR* in lung cancer samples via western blotting overlays.

5

Patient

22803

Tumor

2805#2

22803

Normal

2D western blot overlays from tumor (left) and normal (right) tissue samples. EGFR

(red) over pTyr (white) after stripping and reprobing same blot. *EGFR = epidermal

growth factor receptor.

2837#3

220

94

60

43

29

basic acidic

Results must be expressed as a number to compare many samples Ultra high-sensitivity ECL western blotting is picky. Quantification is not trivial.

6

Enhanced Chemiuminescent (ECL) Western Blotting

7

Figue 1 is taken from Amersham ECL Western Blotting detection reagents and analysis system,

Product Booklet Code RPN2106/8/9. (GE Healthcare) HRP = horse radish peroxidase.

First, proteins are

transferred from a 2D

gel to a paper-like

membrane, Hybond

ECL or PVDF.

The ECL light reaction peaks at about 15 min. The darkness of a film pattern depends on where you are in the curve.

8

Taken from Amersham ECL Western Blotting detection reagents and analysis system, Product Booklet

Code RPN2106/8/9. (GE Healthcare) ECL light peaks at about 15 minutes.

Results within a given ECL WB are quantitative. The problem is comparing between blots.

We decided to add a dot blot standard curve strip to every 2D western blot. If the plot showed linearity, results could be normalized relative to one of the dots.

9

Tumor Normal

Optimizing the dot blots wasn’t trivial.

• HRP-secondary antibody gave linear dot blot plots, but

wasn’t stable. Various conditions had to be worked out.

• Finally, the dot blots were standardized. All following

results are from dot blots loaded with a mouse/rabbit IgG

mixture.

ng/dot:

12

8

4

2

1

0.5

0.1

10

Amersham Hyperfilm (GE Healthcare) versus

Kodak Biomax (Sigma)

We use both. Which is better for quantification?

Test method

• Ran 4 pairs of Tumor/Normal samples in duplicate (8 2D gels x 2)

Western blotted one set with Hyperfilm and the other with Kodak.

Included identical dot blots on all.

• Scanned films with our calibrated laser densitometer

- linear from 0 – 3 OD.

• Analyze with SameSpots software (TotalLab, UK.)

11

Examples: Hyperfilm versus Kodak Biomax

12

Hyperfilm,10 min (2901#14) Kodak, 10 min (2908#3)

R² = 0.9838

0

1,000,000

2,000,000

3,000,000

4,000,000

5,000,000

0 2 4 6 8 10 12 14

Int

De

nsi

ty S

S/

La

ser/

Ko

da

k

ng IgG

2908#3, 10 min

R² = 0.7581

0

2,000,000

4,000,000

6,000,000

8,000,000

0 2 4 6 8 10 12 14

Int

De

nsi

ty S

S/La

we

rHyp

er

ng IgG

2901#14, 10min

Dot blot results:

Kodak Biomax film is better for quantification. Reasonable criteria for

acceptance: R2 > 0.95 for dot blot.

13

Sample

Gel ID,

exposure time

Dot blot

R2 Sample

Gel ID,

exposure time

Dot blot

R2

2908#1, 3 min 0.9917

2908#1, 10 min 0.9830

2908#2, 3 min 0.9925

2908#2, 10 min 0.9356

2908#3, 3 min 0.9917

2908#3, 10 min 0.9838

2908#4, 3 min 0.9689

2908#4, 10 min 0.9856

2901#12, 3 min 0.9618 2908#5, 3 min 0.9163

2901#12, 10 min 0.8695 2908#5, 10 min 0.9789

2901#13, 3 min 0.9639 2908#6, 3 min 0.9838

2901#13, 10 min 0.9185 2908#6, 10 min 0.9739

2901#14, 3 min 0.9488 2908#7, 3 min 0.9977

2901#14, 10 min 0.7581 2908#7, 10 min 0.9534

2901#15, 3 min 0.9429 2908#8, 3 min 0.9740

2901#15, 10 min 0.6965 2908#8, 10 min 0.9677

0.8825 0.9737

31026 T 22803 T

31026 N 22803 N

Kodak average R2 Hyperfilm average R2

30934 N 31026 N

31102 T 31102 T

31102 N 31102 N

Optimization

30417 T 30417 T

30417 N 30417 N

30934 T 31026 T

Amersham ECL Hyperfilm Kodak Biomax MR

Conditional

formatting:

R2 < 0.95 = red

Analysis of human lung tumor samples

Consider:

1. Protein patterns from tumors are

different than those from matched

normal tissue. The latter is bloodier in

this case.

2. We’re trying to quantify low-abundance

TKs. High abundance proteins will be

ignored.

14

T1 N1 T2 N2 T3 N3 T4 N4

Albumin from residual blood is

higher in normal samples

Four matched pairs of human squamous cell

carcinoma. T = tumor, N = normal tissue.

High abundance proteins are visible on the Coomassie-stained PVDF blot

15

1. Cover ECL film with transparency.

2. Align film with printed PVDF Coomassie image using corner marks.

3. Outline Coomassie-stained proteins in red on transparency.

4. Outline low abundance protein spots unique to film in blue (8, 9 & 10 above.)

5. Analyze low abundance proteins in all samples using SameSpots software.

Coomassie-stained PVDF WB film exactly matches Coomassie PVDF

North star

spot, in all

samples

Quantitative Analysis using SameSpots software

Analysis Steps

1. Scan the films with a calibrated laser densitometer.

2. Align the 2D gel images on the computer screen.

3. Hand outline and match spots of interest.

4. Subtract background, normalize spot densities,

create montages.

5. Export data to Excel.

16

Eight pTyr spots of interest were outlined in every image.

Examples of two matched pTyr western blots pairs: 22803 tumor (top left), 22803 normal (bottom left); 31026

tumor (top right), 31026 normal (bottom right.) Spot numbering is shown in the upper left image only. Spot 11

is the “North Star”. If abundant proteins are disallowed, only a few proteins differ between tumor and matched

normal tissue. The EGFR 175 kDa protein is not present in the 31026 film, but a different protein at ~30 kDa is

strongly lighting up. 17

22803 T 31026 T

22803 N 31026 N

21 (EGFR)

14

15

16

11 10

18 19

Isoforms of same protein?

Quantitative Results from SameSpots software.

Table 1. Raw data from SameSpots software. Integrated density within a spot outline normalized

as a percent of the 2 ng spot on the dot blot for that film.

18

2908#05

31102-T

3 min

2908#05

31102-D

10 min

2908#06

31102-N

3 min

2908#06

31102-N

10 min

2908#07

22803-T

3 min

2908#07

22803-T

10 min

2908#08

22803-N

3 min

2908#08

22803-N

10 min

2908#01

30417-T

3 min

2908#01

30417-T

10 min

2908#02

30417-N

3 min

2908#02

30417-N

10 min

2908#03

31026-T

3 min

2908#03

31026-T

10 min

2908#04

31026-N2

3 min

2908#04

31026-N

10 min

ng IgG Norm. Vol. Norm. Vol. Norm. Vol. Norm. Vol. Norm. Vol. Norm. Vol. Norm. Vol. Norm. Vol. Norm. Vol. Norm. Vol. Norm. Vol. Norm. Vol. Norm. Vol. Norm. Vol. Norm. Vol. Norm. Vol.

12 1,914.2 702.2 833.7 383.3 991.5 421.8 669.7 321.7 921.6 496.8 720.7 339.9 602.8 341.3 854.6 530.5

8 711.7 361.0 440.7 264.8 654.0 330.6 335.0 189.3 194.5 165.3 516.7 321.6 404.0 268.9 432.5 328.9

4 257.0 195.9 265.0 190.8 322.8 233.0 172.0 140.0 232.2 213.0 216.6 176.8 165.0 156.3 256.7 225.3

2 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0

1 30.5 40.1 38.1 46.0 22.0 27.9 40.2 46.1 30.5 38.4 30.4 32.9 29.3 40.4 33.6 35.3

0.5 25.8 34.3 15.7 19.4 11.4 15.8 19.0 23.3 14.7 17.8 40.2 38.7 22.4 29.6 51.1 50.7

0.1 - 4.3 6.5 6.1 - 3.6 9.5 12.3 - 2.8 18.1 18.7 27.4 34.8 25.0 27.1

Spot #

10 28.2 18.6 8.6 3.5 441.7 237.1 4.7 1.9 38.2 31.6 5.4 4.3 434.6 210.2 8.6 6.9

11 330.7 158.3 41.5 34.0 219.6 126.4 108.3 71.6 878.0 391.0 472.7 200.1 402.1 198.5 461.3 286.8

14 174.8 68.4 48.5 21.4 0.5 0.4 32.5 22.6 266.7 110.6 1.3 2.5 0.4 0.3 105.4 39.0

15 33.4 21.4 25.9 10.0 2.3 5.6 17.1 11.0 107.4 50.4 162.4 54.7 5.3 4.2 38.5 32.7

16 2.4 2.8 5.8 5.3 0.6 0.5 10.2 9.3 21.2 14.0 9.5 4.6 1.2 0.8 11.0 8.3

18 15.8 3.5 7.6 2.8 7.3 2.6 4.9 1.4 899.0 428.0 9.6 5.3 10.6 11.7 12.7 3.7

19 112.6 84.3 3.7 0.9 3.1 1.0 2.3 0.6 7,360.2 3,012.2 4.3 2.7 3.2 1.1 4.2 2.5

21 71.9 46.0 48.3 52.8 594.3 473.0 28.4 34.3 210.0 159.4 102.9 45.4 37.6 47.9 36.0 13.5

Comparison of 3 & 10 min film exposures

Table 2. Tumor/normal ratios for two film exposures. T/N ratio >10, red; < 0.1, green.

Ideally, the two film exposures would give the same value. *R2 values for dotblots < 0.95.

• There is fairly good agreement between 3 and 10 min exposures.

• Setting the red/green cutoff to detect > 10-fold changes highlights the same spot

regardless of film exposure.

19

31102

T/N

31102

T/N

22803

T/N

22803

T/N

30417

T/N

30417

T/N

31026

T/N

31026

T/N

Spot # MW 3 min* 10 min 3 min 10 min 3 min 10 min* 3 min 10 min

21 175 kDa 1.5 0.9 21 14 2.0 3.5 1 4

14 60 kDa 3.6 3.2 0.01 0.02 211 44 0.004 0.01

15 60 kDa 1.3 2.1 0.1 0.5 0.7 0.9 0.14 0.13

16 60 kDa 0.4 0.5 0.1 0.1 2.2 3.0 0.11 0.10

18 30 kDa 2.1 1.3 1.5 1.9 94 81 0.8 3.2

10 30 kDa 3.3 5.4 93 125 7.1 7.4 51 30

19 30 k Da 30 90 1.3 1.7 1700 1129 0.8 0.4

11 32 kDa 8.0 4.7 2.0 1.8 1.9 2.0 0.9 0.7

2908 #5* & 6 2908# 7&8 2908 #1&2* 2908 #3&4

Final Report: Does it reflect actual differences in films?

Table 2. Fold change (Tumor/Normal ratios) for eight pTyr proteins in four samples.

The 3 and 10 min exposure values were averaged. Red: ratio > 10; Green: ratios <

0.1.

20

Patient #

S21,

EGFR

S18,

30-A

S10,

30-B

S19,

30-C

S14,

60-X

S15,

60 -Y

S16,

60-Z

S11

32-marker

31102 1 2 4 60 3 2 0.5 6

22803 17 2 109 2 0.02 0.3 0.05 2

30417 3 87 7 1414 127 0.8 2.6 2

31026 2 2 41 1 0.01 0.1 0.1 1

pTyr-Protein Fold Increase: Tumor/Normal

Spot 21, EGFR

175 kDa

Patient 22803

Tumor

Normal

Spots 19, 10, & 18

Table 2. Fold change (Tumor/Normal ratios) for eight pTyr protein spots in four

samples. Red: T/N ratio > 10; Green: T/N ratio < 0.1.

21

19 10 31102

18

22803

30417

31026

19 10 18

Tumor Normal Lung

Patient #

S21,

EGFR

S19,

30-C

S10,

30-B

S18,

30-A

S14,

60-X

S15,

60 -Y

S16,

60-Z

S11

32-marker

31102 1 60 4 2 3 2 0.5 6

22803 17 2 109 2 0.02 0.3 0.05 2

30417 3 1414 7 87 127 0.8 2.6 2

31026 2 1 41 2 0.01 0.1 0.1 1

pTyr-Protein Fold Increase: Tumor/Normal

Spots 14 &16 are much darker in normal tissue for 2 patients.

Table 2. Fold change (Tumor/Normal ratios) for eight pTyr proteins in four samples.

Red: T/N ratio > 10; Green: T/N ratios < 0.1.

22

14

15

16

22803 30417 31026

Tumor Normal Tumor Normal Tumor Normal

Patient #

S21,

EGFR

S19,

30-C

S10,

30-B

S18,

30-A

S14,

60-X

S15,

60 -Y

S16,

60-Z

S11

32-marker

31102 1 60 4 2 3 2 0.5 6

22803 17 2 109 2 0.02 0.3 0.05 2

30417 3 1414 7 87 127 0.8 2.6 2

31026 2 1 41 2 0.01 0.1 0.1 1

pTyr-Protein Fold Increase: Tumor/Normal

Conclusions: 1. Numerical results presented in the final table show

good agreement with differences observed visually on

the ECL films. This preliminary work suggests that

protein differences in 2D gel western blots can be

expressed numerically.

2. The dot blot standard curves provide criteria for

quantitative comparisons.

23

Future work: 1. Validate the method to determine within-day and

between-day variability.

Acknowledgements

24

Jon Johansen

Lab Manager

Matt Hoelter

Western Blot Manager

Andrew Koll, Biochemist

RTK mechanism, inspiration

The Biology of Cancer

by Robert Weinberg

Publisher: Garland Science

Second Edition 2013

Kendrick Collaborators:

Thanks to the 2D software

developers at TotalLab

http://www.totallab.com